The invention relates generally to spiro-[pyrrolidine-2,3xe2x80x2-oxindole] compounds, to combinatorial libraries of spiro[pyrrolidine-2,3xe2x80x2-oxindole] compounds, and to methods of synthesizing and assaying such libraries. The compounds can be formed, for example, via 1,3-dipolar cycloaddition of reactive isatin-amino acid adducts to substituted trans-chalcones and other dipolarophiles.
Oxindole alkaloids are a rich class of bioactive compounds. For example, gelsemine is a spirooxindole alkaloid that possesses central nervous system (CNS) stimulating activity. Other spirooxindoles are aldose reductase inhibitors and are used as antidiabetic drugs.
In classical drug design, many individual compounds are synthesized one at a time and then screened. This is a relatively labor-intensive process. An alternative approach is rational drug design. One aspect of rational drug design includes structure-guided methods. One structure-guided approach to the discovery of new pharmaceutically active organic drugs (e.g., compounds with the three-dimensional structure needed for binding) relies primarily on X-ray crystallography of purified receptors. Once a binding site is identified, organic molecules are designed to fit the available steric space and charge distribution. However, it is often difficult to obtain purified receptors, and still more difficult to crystallize the receptor so that X-ray crystallography can be applied.
Other methods such as homology modelling or nuclear magnetic resonance studies can also be used to identify the binding site, although it is still difficult to devise an appropriate ligand, even after the binding site has been properly identified. Overall, it is quite difficult to design useful pharmaceutically active compounds because of factors such as the difficulty in identifying receptors, purifying and identifying the structures of compounds which bind to those receptors, and thereafter synthesizing those compounds.
Another approach to the discovery of new drugs is through pharmacophore-guided design. If a number of molecules (e.g., biologically active compounds) are known to bind, for example, to a macromolecule, new compounds can be synthesized that mimic the known molecules. However, since the active moiety or active structural component of the active compound is usually unknown, the process of synthesizing new compounds relies primarily on trial and error and the synthesis and screening of each compound individually. This method is time consuming and expensive since the likelihood of success for any single compound is relatively low.
Rather than trying to determine the particular three-dimensional structure of a protein using crystallography or attempting to synthesize specific compounds that mimic a known biologically active compound, researchers have also developed assays to screen combinatorial libraries of candidate compounds. More specifically, those attempting to create biologically active compounds produce extremely large numbers of different compounds at the same time either within the same reaction vessel or in separate vessels. The synthesized combinatorial library is then assayed and active molecules are isolated (e.g., in the case of mixtures of compounds) and analyzed.
In general, the invention is based on the discovery that under the right conditions, variously substituted isatins, xcex1-amino acids, and dipolarophiles (e.g., trans-chalcones, acrylate esters, or vinyl oxindoles) can stereo-and regio-selectively react to form libraries of spiro[pyrrolidine-2,3xe2x80x2-oxindole] compounds. The new libraries can be assayed using any of many known screening procedures for activity, e.g., biological activity. For example, the libraries can be screened for activity as drugs (e.g., anticancer drugs, antibiotics, antiviral drugs, antiinflammatory drugs, analgesics, kinase inhibitors, immunomodulators, neuroleptics, sedatives, stimulants, or diagnostic aids), bioseparation agents (e.g., affinity ligands), or pesticides (e.g., herbicides, insecticides, or rodenticides).
In one embodiment, the invention features a method of synthesizing a library of compounds (e.g., including 10, 100, 5,000, 10,000, 100,000 or more compounds). The method includes reacting a plurality of isatins with a plurality of xcex1-amino acids, independently, to form azomethine ylide compounds; and reacting the azomethine ylides with a plurality of dipolarophiles (e.g., chalcones, acrylate esters, vinyl oxindoles, fumarates, maleates, maleimides, cinnamonitriles, nitroolefins, acrylonitriles, vinyl sulfones, or vinyl sulfoxides), independently, to form the library of compounds.
The chalcones can be prepared, for example, by reacting each of a plurality of arylaldehydes, independently, with each of a plurality of acetophenone compounds. Certain acrylate esters (e.g., cinnamates) can be prepared, for example, by reacting each of a plurality of arylaldehydes with trimethylphosphonoacetate under Horner-Emmons condensation reaction conditions. Vinyloxindoles can be prepared, for example, by reacting oxindoles with arylaldehydes, or by reacting isatins with acetophenone compounds. Azomethine ylides can be prepared in situ in the presence of the dipolarophiles.
In certain cases, the library of compounds is prepared in a single compound-per-well format, wherein each well (e.g., a well of a 96-well plate, a test tube, a centrifuge tube, a flask, a beaker, or other container) contains predominantly a single member of a library of the invention.
In another embodiment, the invention features a chemical library that includes ten or more different compounds, each compound being produced from a reaction of each of a plurality of isatins with each of a plurality of xcex1-amino acids, and with each of a plurality of dipolarophiles (e.g., in a [2+3] reaction as shown in FIG. 2). Each of the ten or more compounds is present in the library in a retrievable and analyzable amount.
The invention also features a chemical library that includes ten or more different compounds each present in a retrievable and analyzable amount. Each compound can be represented by the structural formula: 
where R1 to R4, independently, can be hydrogen, alkyl, aryl, carbocyclic, fluoro, chloro, bromo, iodo, thio, hydroxyl, alkylthio, alkoxy, carboxy, sulfonyl, nitro, cyano, or amido groups, or, if compatible with the reaction conditions, keto, formyl, or amino groups or other substituents. R5 to R12, independently, can be hydrogen, alkyl, aryl, or carbocyclic groups. In some cases, R6 (or R7) and R12, R8 and R9, R10 and R11, or R8 (or R9) and R10 (or R11) can together form at least part of a ring. Preferably, at least one of R8 to R11 is an electron withdrawing group.
In another aspect, the invention features a method for identifying a compound that binds to a macromolecule. The method includes screening any of the above libraries for a characteristic that indicates bioactivity. For example, the compound can be a bioactive molecule (i.e., a molecule that affects the function of a target or that modulates the biological activity of a target, by, for example, upregulating or downregulating activity). The compound can also bind to a receptor or inhibit an enzyme.
In still another embodiment, the invention features a method for preparing a spiro[pyrrolidine-2,3xe2x80x2-oxindole] compound. The method includes reacting an isatin (e.g., an isatin of Table 1) with an xcex1-amino acid (e.g., an xcex1-amino acid of Table 2) to form an azomethine ylide; and reacting the azomethine ylide with a chalcone (e.g., a chalcone prepared from the reaction of an arylaldehyde of Table 3 and an acetophenone compound of Table 4) to form the spiro[pyrrolidine-2,3xe2x80x2-oxindole].
In another aspect, the invention features a spiro compound comprising the formula: 
where Ar1 and Ar2 can be, independently, substituted or unsubstituted aryl or heteroaryl groups; R1 to R4, independently, can be hydrogen, alkyl, aryl, carbocyclic, fluoro, chloro, bromo, iodo, thio, hydroxyl, alkylthio, alkoxy, carboxy, sulfonyl, nitro, cyano, or amido groups, or, if compatible with the reaction conditions, keto, formyl, or amino groups or other substituents; and R5 to R8 and R12, independently, can be hydrogen, alkyl, aryl, or carbocyclic groups. In some cases, R6 (or R7) and R12 can together form at least part of a ring.
An xe2x80x9carylaldehydexe2x80x9d is a compound having the following general structural formula: 
wherein R is covalently bound to the carbon atom. The xe2x80x9cRxe2x80x9d can be any aromatic group (i.e., phenyl or substituted phenyl) or heteroaromatic group (e.g., furyl or pyridyl or substituted variants thereof).
An xe2x80x9cacetophenone compoundxe2x80x9d is a compound having the following general structural formula: 
wherein R is covalently bound to the carbon atom. The xe2x80x9cRxe2x80x9d can be any aromatic or heteroaromatic group. Examples of acetophenone compounds include acetophenone, propiophenone, butyrophenone, and other acetophenone compounds listed in Table 4.
xe2x80x9cChalconesxe2x80x9d are compounds having two aryl groups conjugated to each other through an xcex1,xcex2-unsaturated ketone. Thus, the parent chalcone has the structure: Ar1xe2x80x94C(xe2x95x90O)xe2x80x94C(H)xe2x95x90C(H)xe2x80x94Ar2. The aryl groups, Ar1 and Ar2, can be any substituted or unsubstituted aromatic or heteroaromatic groups.
A xe2x80x9clibraryxe2x80x9d is a collection of compounds (e.g., as a mixture or as individual compounds) synthesized from various combinations of two or more starting components (i.e., a combinatorial library). At least some of the compounds must differ from at least some of the other compounds in the library. A library can, for example, include 5, 10, 50, 100, 1,000, 10,000, 50,000, 100,000 or more different compounds (i.e., not simply multiple copies of the same compounds, although some compounds in the library may be duplicated or represented more than once). Each of the different compounds, whether they have a different basic structure or different substituents, will be present in an amount such that its presence can be determined by some means, e.g., can be isolated, analyzed, and detected with a receptor or suitable probe. The actual quantity of each different compound needed so that its presence can be determined will vary due to the actual procedures used and may change as the technologies for isolation, detection, and analysis advance. When the compounds are present in a mixture in substantially equimolar amounts, for example, an amount of 100 picomoles of each compound can often be detected. Preferably, the average purity of the compounds in the libraries of the invention is at least 70%, 85%, 90%, 95%, 99%, or higher.
Libraries can include both libraries of individual compounds (e.g., present substantially as a single compound-per-well, e.g., made via parallel synthesis) and mixtures containing substantially equimolar amounts of each desired compound (i.e., wherein no single compound dominates or is completely suppressed in any assay). Either library format can allow identification of an active compound discovered in an assay. Spatially arranged (or spatially addressable) array formats (see, e.g., U.S. Ser. No. 09/061,572, filed Apr. 16, 1998, and U.S. Pat. No. 5,712,171) can also be used to develop structure-activity relationships (SARs).
In this description, a xe2x80x9ccompoundxe2x80x9d can be a cyclic or an acyclic molecule, including, for example, carbon, hydrogen, nitrogen, and oxygen atoms, and possibly one or more other heteroatoms, including sulphur, phosphorus, halogens, metals, or other substituents.
Substituents on the organic compound can include one or more carbon, oxygen, hydrogen, iodine, bromine, chlorine, fluorine, nitrogen, sulfur, phosphorus, metal atoms, or any combination of these or other atoms. Typically, substituents can be attached to the isatins, amino acids, and dipolarophiles, and can include alkyls, alkenyls, alkynyls, and aryls, each of which may be either unsubstituted or substituted (e.g., as esters, carboxylic acids, nitriles, ethers, amides, and possibly aldehydes, ketones, or amines, where compatible with the reaction conditions), and may be cyclic, polycyclic, heterocyclic, or acyclic. The general structure of each of these groups is well known. Substituents can also be drawn from any other groups that can be bonded to an organic compound, for example, via a carbon, oxygen, or nitrogen atom.
A non-limiting list of examples of substituents includes hydrogen, hydroxy, Ra, xe2x80x94ORa, xe2x80x94NRaRb, xe2x80x94SO1,2,3,4Ra, xe2x80x94C(O)Ra, xe2x80x94C(O)ORa, xe2x80x94OC(O)Ra, xe2x80x94OC(O)ORa, xe2x80x94NRbC(O)Ra, xe2x80x94C(O)NRaRb, xe2x80x94OC(O)NRaRb, xe2x80x94NRcC(O)NRaRb, xe2x80x94NRbC(O)ORa, xe2x80x94Raxe2x80x94Oxe2x80x94Rb, xe2x80x94RaNRbRc, xe2x80x94Raxe2x80x94Sxe2x80x94Rb, xe2x80x94Raxe2x80x94S(O)xe2x80x94Rb, xe2x80x94Raxe2x80x94S(O))2xe2x80x94Rb, xe2x80x94ORaxe2x80x94Oxe2x80x94Rb, xe2x80x94NRaRbxe2x80x94Oxe2x80x94Rc, xe2x80x94SO1,2,3,4Raxe2x80x94Oxe2x80x94Rb, xe2x80x94C(O)Raxe2x80x94Oxe2x80x94Rb, xe2x80x94C(O)ORaxe2x80x94Oxe2x80x94Rb, xe2x80x94OC(O)Raxe2x80x94Oxe2x80x94Rb, xe2x80x94OC(O)ORaxe2x80x94Oxe2x80x94Rb, xe2x80x94NRbC(O)Raxe2x80x94Oxe2x80x94Rc, xe2x80x94C(O)NRaRbxe2x80x94Oxe2x80x94Rc, xe2x80x94OC(O)NRaRbxe2x80x94Oxe2x80x94Rc, xe2x80x94NRcC(O)NRaRbxe2x80x94Oxe2x80x94Rd, xe2x80x94NRbC(O)ORaxe2x80x94Oxe2x80x94Rc, xe2x80x94ORaxe2x80x94Sxe2x80x94Rb, xe2x80x94NRaRbxe2x80x94Sxe2x80x94Rc, xe2x80x94SO1,2,3,4Raxe2x80x94Sxe2x80x94Rb, xe2x80x94C(O)Raxe2x80x94Sxe2x80x94Rb, xe2x80x94C(O)ORaxe2x80x94Sxe2x80x94Rb, xe2x80x94OC(O)Raxe2x80x94Sxe2x80x94Rb, xe2x80x94OC(O)ORaxe2x80x94Sxe2x80x94Rb, xe2x80x94NRbC(O)Raxe2x80x94Sxe2x80x94Rc, xe2x80x94C(O)NRaRbxe2x80x94Sxe2x80x94Rc, xe2x80x94OC(O)NRaRbxe2x80x94Sxe2x80x94Rc, xe2x80x94NRcC(O)NRaRbxe2x80x94Sxe2x80x94Rd, xe2x80x94NRbC(O)ORaxe2x80x94Sxe2x80x94Rc, xe2x80x94ORaxe2x80x94NRbRd, xe2x80x94NRaRbxe2x80x94NRcRd, xe2x80x94SO1,2,3,4Raxe2x80x94NRbRd, xe2x80x94C(O)Raxe2x80x94NRbRd, xe2x80x94C(O)ORaxe2x80x94NRbRd, xe2x80x94OC(O)Raxe2x80x94NRbRd, xe2x80x94OC(O)ORaxe2x80x94NRbRd, xe2x80x94NRbC(O)Raxe2x80x94NRcRd, xe2x80x94C(O)NRaRbxe2x80x94NRcRd, xe2x80x94OC(O)NRaRbxe2x80x94NRcRd, xe2x80x94NRcC(O)NRaRbxe2x80x94NHRd, and xe2x80x94NRbC(O)ORaxe2x80x94NRcRd; where Ra, Rb, Rc, and Rd are each independently alkyl, alkenyl, alkynyl, aryl, aralkyl, aralkenyl, or aralkynyl groups having, e.g., 1 to 6, 10, 20, or even 30 carbon atoms. Ra, Rb, Rc and Rd can each be substituted, for example, with halo (e.g., 1 to 6 halogen atoms), nitro, hydroxyl, alkyl (e.g., having 1 to 6 carbon atoms), mercapto, sulfonyl, nitro, cyano, amino, acyl, acyloxy, alkylamino, dialkylamino, trihalomethyl, nitrilo, nitroso, alkylthio, alkylsulfinyl, or alkylsulfonyl. The substituents can include electron withdrawing groups, electron donating groups, Lewis acids, Lewis bases, as well as polar, nonpolar, hydrophilic, and hydrophobic functional groups.
An electron withdrawing group is a moiety that is capable of decreasing electron density in other parts of a compound to which it is covalently attached. Non-limiting examples of electron withdrawing groups useful in the invention include nitro, carbonyl, cyano, iodo, bromo, chloro, fluoro, and sulfone groups.
An electron donating group is a moiety that is capable of increasing electron density in other parts of a compound to which it is covalently attached. Non-limiting examples of electron donating groups useful in the invention include alkyl, amine, hydroxyl, and alkoxy.
Examples of substituents also include aliphatic (e.g., alkyl or alkenyl), alicyclic (e.g., cycloalkyl or cycloalkenyl), or aromatic (e.g., phenyl or naphthyl) substituents, aliphatic and alicyclic-substituted aromatic nuclei (e.g., p-(n-butyl)-phenyl or o-xylyl), as well as cyclic substituents wherein the ring is completed through another portion of the molecule (i.e., for example, any two indicated substituents can together form an alicyclic radical).
Hetero substituents are also contemplated. These are substituents that contain an atom or atoms other than carbon in a ring or chain otherwise composed of carbon atoms. Suitable heteroatoms include, for example, sulfur, oxygen, and nitrogen. Hetero substituents therefore include groups such as epoxides, ethers, pyridines, piperazines, furans, pyrrolidines, and imidazoles.
xe2x80x9cAlkyl groupsxe2x80x9d should be construed to include both linear chain and branched chain derivatives of any substituted or unsubstituted acyclic carbon-containing moieties, including alkanes, alkenes, and alkynes. Alkyl groups having one to five, ten, twenty, or even more carbon atoms are possible. Examples of alkyl groups include lower alkyls, for example, methyl, ethyl, n-propyl, iso-propyl, n-butyl, iso-butyl, tert-butyl; higher alkyls, for example, octyl, nonyl, and decyl; lower alkenyls, for example, ethenyl, propenyl, propadienyl, butenyl, butadienyl; higher alkenyls such as 1-decenyl, 1-nonenyl, 2,6-dimethyl-5-octenyl, and 6-ethyl-5-octenyl; and alkynyls such as 1-ethynyl, 2-butynyl, and 1-pentynyl. Other linear and branched alkyl groups are also within the scope of the present invention.
In addition, such alkyl groups can also contain various substituents in which one or more hydrogen atoms has been replaced by a functional group. Functional groups include, but are not limited to, tertiary amine, amide, ester, ether, and halogen, i.e., fluorine, chlorine, bromine and iodine. Specific substituted alkyl groups can be, for example, alkoxy such as methoxy, ethoxy, butoxy, and pentoxy; dimethylamino, diethylamino, cyclopentylmethyl-amino, benzylmethylamino, and dibenzylamino; formamido, acetamido, or butyramido; methoxycarbonyl or ethoxycarbonyl; or dimethyl or diethyl ether groups.
xe2x80x9cCarbocyclic groupsxe2x80x9d include both substituted and unsubstituted, cyclic, carbon-containing moieties such as cyclopentyl, cyclohexyl, cycloheptyl, and adamantyl. Such cyclic groups can also contain various substituents in which one or more hydrogen atoms have been replaced by a functional group. Such functional groups include those described above, as well as lower alkyl groups as described above. The cyclic groups of the invention can also include one or more heteroatoms, for example, to form heterocyclyls.
xe2x80x9cAryl groupsxe2x80x9d include substituted and unsubstituted hydrocarbon rings bearing a system of conjugated double bonds, usually comprising (4n+2) pi bond electrons, where n is zero or a positive integer. Examples of aryl groups include, but are not limited to, phenyl, naphthyl, anisyl, tolyl, xylyl and the like. Aryl groups can also include aryloxy, aralkyl, aralkyloxy and heteroaryl groups, e.g., pyrimidine, morpholine, piperazine, piperidine, benzoic acid, toluene, thiophene, and the like. These aryl groups can also be substituted with any number of a variety of functional groups. In addition to the functional groups described above in connection with substituted alkyl groups and carbocyclic groups, functional groups on the aryl groups can also include other nitrogen, oxygen, sulfur, or halogen bearing groups.
It is to be understood that this invention is not limited to the particular compounds and their substituents described herein; such compounds and their substituents, as well as the methods used in their manufacture and use, can, of course, vary. Also, many of the compounds of the new libraries, compounds produced by the new methods, and compounds shown in the figures can exist in two or more stereoisomeric forms. Unless specifically stated otherwise herein, the invention should be understood to include all stereoisomeric permutations of these compounds.
Throughout this description and the claims, it must be noted that the singular forms xe2x80x9ca,xe2x80x9d xe2x80x9can,xe2x80x9d and xe2x80x9cthexe2x80x9d include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to xe2x80x9can organoboronic acidxe2x80x9d or xe2x80x9ca benzylic aminexe2x80x9d includes groups or subgroups of organoboronic acids or benzylic amines. Similarly, reference to xe2x80x9csolventxe2x80x9d includes reference to mixtures of solvents, and reference to xe2x80x9cthe methodxe2x80x9d includes a plurality of methods.
The present invention includes a variety of different aspects, including novel acyclic, cyclic, and heterocyclic organic compounds, libraries of such compounds, and processes for synthesizing such compounds and libraries thereof. Further, within each of these aspects of the invention, the present invention includes a number of specific embodiments. The invention provides processing technology to produce and isolate compounds and libraries of compounds, one or more of which can mimic the activity or characteristics of naturally-occurring molecules or synthetic biologically active molecules, but which compounds can have different chemical structures as compared to the natural molecule or synthetic molecule. The word xe2x80x9cmimicxe2x80x9d is used loosely, in that the compounds produced can have the same activity, greater activity, or lesser activity than naturally occurring molecules or biologically active synthetic molecules, or can block the activity of these molecules entirely. Furthermore, the compounds can have similar or radically different structures compared to naturally occurring molecules.
The methods described herein can be used to create us libraries of compounds that differ from the specific libraries described below, but which are also within the scope of the invention.
The term xe2x80x9cdipolarophilexe2x80x9d is used herein to describe any compound that can react with an azomethine ylide in a [3+2] fashion. For example, activated olefins (e.g., olefins substituted with at least one electron withdrawing group, such as chalcones, acrylates, fumarates, maleates, maleimides, cinnamates, cinnamonitriles, nitroolefins, acrylonitriles, vinyl sulfones, vinyl oxindoles, and vinyl sulfoxides) can be suitable dipolarophiles.
xe2x80x9cIsatinsxe2x80x9d are cyclic xcex1-ketoamides having an aryl group to which a five-member ring is fused. xe2x80x9cFusedxe2x80x9d rings have two atoms in common. In an isatin, two of the remaining three ring members are the carbon atoms of carbonyl ( greater than Cxe2x95x90O) groups, and the third remaining member is the nitrogen of the amide. In general, isatins have the structure: 
where R1 to R4, independently, can be hydrogen, alkyl, aryl, carbocyclic, fluoro, chioro, bromo, iodo, thio, hydroxyl, alkylthio, alkoxy, carboxy, sulfonyl, nitro, cyano, or amido groups, or, if compatible with the reaction conditions, is keto, formyl, or amino groups or other substituents; and R5 can be hydrogen, alkyl, aryl, or carbocyclic groups.
xe2x80x9cxcex1-Amino acidsxe2x80x9d include a carboxylic acid group (xe2x80x94C(xe2x95x90O)xe2x80x94OH) and an amino group (xe2x80x94N(R13)(R14); where R13 and R14 can be, independently, hydrogen or another substituent), separated by a single methylene moiety (xe2x80x94C(R15)(R16)xe2x80x94; where R15 and R16 can be, independently, hydrogen or another substituent).
A xe2x80x9cspiro compoundxe2x80x9d is an organic compound or moiety that has a structure including two closed rings where the two rings have a single carbon atom in common with each other. The compound can be saturated or unsaturated. A spiro compound can be mono-, bi-, tri-, or polycyclic depending on the number of rings present; the three major groups of cyclic compounds include: (1) alicyclic, (2) aromatic (also called arene) and (3) heterocyclic. Spiro[pyrrolidine-2,3xe2x80x2-oxindole] is a spiro compound.
A xe2x80x9cretrievable amountxe2x80x9d is an amount of a compound in a library that is present in a concentration such that the compound can be separated from the other compounds of the library (i.e., in the case of mixtures) by standard techniques. Preferably, at least 50 or 100 pmol, of a compound is present in a library when the compounds of the library are present in approximately equimolar amounts.
An xe2x80x9canalyzable amountxe2x80x9d is an amount of a compound that is present in a library such that the compound can be detected and identified in the library. At least approximately 10 pmol, more preferably 50 pmol of a compound should be present in the library when the components of the library are present in approximately equal molar amounts.
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described below. All publications, patent applications, patents, technical manuals, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present application, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.
Advantages of the new methods and libraries include incorporation of a plurality of diversity elements in a single step. A diversity element is any part of a compound that varies among the members of a library. The amino acid components can introduce at least two diversity elements, for example, one at the xcex1-side chain and one as a substituent on the amino group. The isatins can also be N-substituted in addition to having a substituent or substituents on the aromatic ring, thus incorporating two diversity elements. Finally, the chalcones can have independent substituents on their two aromatic rings, also introducing up to two diversity elements.
It should be noted that not all of these diversity elements are varied in every library (or member of the library) of the invention. For example, a library can be made wherein the substitution at the nitrogen of the isatin is constant in all the members in the library. Additional diversity elements can also be introduced; for example, the aryl groups of the chalcone can be based on a variety of heteroaromatic groups (each of which can be substituted) rather than simply being a phenyl group.
The new libraries allow exploration of structure-activity relationships based on the interactions between the compounds in the library and natural receptor sites.
Another advantage of the present invention is that the new methods can allow rapid, simultaneous synthesis of a vast number of independent compounds in greater than 50%, 75%, or even 85% yield in some cases. Such high yields, although not a critical feature of the present libraries and methods, can allow preparation of libraries of substantially pure individual compounds without the need for extensive purification (e.g., a single compound-per-well). For certain applications, impurities can be highly detrimental. For example, agrichemicals are often used in large quantities and a substantial impurity in such chemicals can have undesired side effects. However, even low yielding reactions or reactions that produce significant amounts of impurities can be used in the new methods (e.g., followed by some purification, or if libraries containing mixtures of compounds are suitable).
In the single compound-per-well format, each well or reaction vessel contains a predominant species. It is not necessary that the predominant species be 100% pure; all that is required is that the predominant species be pure enough that structure-activity relationships can be reliably probed in a primary screen without the need for additional deconvolution. Although in some cases, the interaction of the predominant species with an impurity can result in false positive or false negative results, a small amount of impurity is often tolerable.
New (i.e., second generation) libraries can be constructed based on the structure-activity relationships derived from a primary screen. Structure-activity relationships found in the second generation screens can in turn serve as the basis for construction of subsequent generations of libraries. Through such an iterative process, it is possible to rapidly implement lead optimization strategies and to confirm or supplant existing theories or assumptions regarding binding (see, e.g., Zambias et al., U.S. Pat. No. 5,712,171). These methods can themselves result in compounds having the same or stronger affinity for a natural receptor site as a natural or known bioactive compound that ordinarily binds the same site. The methods can also result in compounds with superior properties relating to absorption, distribution, metabolism, toxicity, or stability.
Pharmaceutically active compounds are often highly substituted heterocycles; there is therefore a need for a method to rapidly synthesize a large number of related substituted heterocyclic compounds quickly and relatively inexpensively. The present methods overcome the problem of a separate synthesis for each member of a group of candidate compounds where the structural components conferring biological activity are unknown.
Other features and advantages of the invention will be apparent from the following detailed description, and from the claims.